Electrophoresis is one of the most widely used separation techniques in the biologically-related sciences. Molecular species such as peptides, proteins, and oligonucleotides are separated by causing them to migrate in a buffer solution under the influence of an electric field. This buffer solution normally is used in conjunction with a low to moderate concentration of an appropriate gelling agent such as agarose or polyacrylamide to minimize the occurrence of convective mixing.
Two primary separating mechanisms exist, separations based on differences in the effective charge of the analytes, and separations based on molecular size. The first of these mechanisms is limited to low or moderate molecular weight materials in the case of separations of oligonucleotides because in the high molecular weight range the effective charges of these materials become rather similar, making it difficult or impossible to separate them. In the case of proteins, charge and size can be used independently to achieve separations. Separations based on molecular size are generally referred to as molecular sieving and are carried out employing as the separating medium gel matrices having controlled pore sizes. In such separating systems, if the effective charges of the analytes are the same, the separation results from differences in the abilities of the different sized molecular species to penetrate through the gel matrix. Smaller molecules move relatively more quickly than larger ones through a gel of a given pore size. Oligonucleotides and medium to high molecular weight polypeptides and proteins are commonly separated by molecular sieving electrophoresis. In the case of proteinaceous materials, however, it is first generally necessary to modify the materials to be separated so that they all have the same effective charges. This is commonly done by employing an SDS derivatization procedure, such as is discussed in "Gel Electrophoresis of Proteins," B. D. Hames and D. Rickwood, Eds., published by IRL Press, Oxford and Washington, D.C., 1981. The contents of this book are hereby incorporated herein by reference.
Sometimes it is desirable to separate proteinaceous materials under conditions which pose a minimal risk of denaturation. In such cases, system additives such as urea and SDS are avoided, and the resulting separations are based on differences in both the molecular sizes and charges.
Most electrophoretic separations are today conducted in slabs or open beds. However, such separations are hard to automate or quantitate. Extremely high resolution separations of materials having different effective charges have been achieved by open tubular free-zone electrophoresis and isotachophoresis in narrow capillary tubes. In addition, bulk flow can be driven by electroosmosis to yield very sharp peaks. Such high efficiency open tubular electrophoresis has not generally been applied to the separation of medium to high molecular weight oligonucleotides, however, since these materials have very similar effective charges, as indicated above. In addition, open tubular electrophoresis does not provide size selectivity for proteinaceous materials.
Hjerten has published an article in the Journal of Chromatography, 270, 1-6 (1983), entitled "High Performance Electrophoresis: The Electrophoretic Counterpart of High Performance Liquid Chromatography," in which he employs a crosslinked polyacrylamide gel in tubes having inside dimensions of 50-300 micrometers, and wall thicknesses of 100-200 micrometers. However, this work suffers from limited efficiency and relatively poor performance due in part to the use of relatively wide bore capillaries, relatively low applied fields, high electrical currents, and insufficient suppression of electroendosmosis. He has also obtained U.S. Pat. No. 3,728,145, in which he discloses a method for coating the inner wall of a large bore tube with a neutral hydrophilic substance such as methyl cellulose or polyacrylamide to reduce electroendosmosis in free-zone electrophoresis in open tubes. In a later patent, U.S. Pat. No. 4,680,201, Hjerten discloses a method for coating the inner wall of a narrow bore capillary with a monomolecular polymeric coating of polyacrylamide bonded to the capillary wall by means of a bifunctional reagent. These capillaries are also open tubes to be used for free-zone electrophoresis.
The work in the field of electrophoresis in capillaries by researchers other than the present applicants has generally resulted in polyacrylamide columns which were not highly stable and could not be subjected to sufficiently high electric fields to achieve high efficiencies and high resolution separations. Improved capillary columns containing a polyacrylamide polymer network for electrophoresis which provide superior stability, and efficiency, have been disclosed by the present inventors in application Ser. No. 07/421,609, (Batch No. P34), the entire contents of which are hereby incorporated by reference. The capillary columns disclosed in the above mentioned Patent Application are suitable for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); however, UV detection of SDS-proteins in polyacrylamide-filled capillaries is strictly limited to a specific wavelength of about 250 nm and higher due to the high UV absorbance associated with polyacrylamide gels, crosslinked or uncrosslinked.
For example, FIG. 1 shows the UV spectra of a 3%T (total monomer) and 0%C (crosslinker) polyacrylamide polymer network polymerized in distilled water. As is seen in FIG. 1, the absorbance is very high at the lower UV range. Moreover, the higher the acrylamide (or bisacrylamide) content of the polymer network, the higher the absorption.
It is known that proteins intensively absorb at 214 nm due to the peptide bonds. The absorption at 214 nm can be called non-specific, since all peptide bonds absorb at that wavelength, while absorption at 280 nm is restricted to the proteins containing aromatic amino acids. FIG. 2 shows a typical UV spectrum of a protein. As is shown in FIG. 2, the intensity of the absorption at 214 nm is much higher than that at 280 nm.
Therefore, the sensitivity and the selectivity of UV detection would be at its highest if measurements could be taken at 214 nm. However, due to the high absorption of the polyacrylamide-filled capillaries at a wavelength of 214 nm, measurements are instead taken at wavelengths of around 280 nm. Thus, although the polyacrylamide-filled capillaries do not exhibit intense absorption at 280 nm, the selectivity and sensitivity for detecting proteins is very reduced at that wavelength.
Therefore, the sensitivity and selectivity of on-column UV detection of SDS-proteins such as is used in a capillary electrophoresis system could be greatly improved if on-column detection was achieved at lower UV wavelengths.